1 – Instrumentation

A type of flow cytometer manufactured and sold by BD Biosciences. This instrument was one of the first mass produced flow cytometers. The FACSCalibur is still prevalent in many labs around the world. While only a four color, six parameter analog system, this machine is stable and rarely requires service. It has gained a reputation as a “work horse” in core facilities. Recently, companies such as Cytek have begun refurbishing these analog instruments into digital models. This hybrid instrument produces a machine capable of acquiring data digitally, in FCS3.0 format, while still utilizing the reliable fluidics system that the Calibur…

PBS is the acronym for phosphate buffered saline. Phosphate buffer is one of the most common buffers used in biological research.  The phosphate serves as a buffer to keep the pH constant, while the saline is referencing the osmolarity.  Additional ions such as Ca2+ or Mg2+ , energy sources like glucose, or chelators such as EDTA can be added based on the specific needs of the experiment. There are many different formulations of PBS, based on the cell type and needs.  The most common, from Cold Spring Harbor is shown below, providing a pH of 7.4 with an osmolarity to normal…

What is autofluorescence? Autofluorescence is the term given to describe the natural fluorescence that occurs in cells. The common compounds that give rise to this fluorescence signal include cyclic ring compounds like NAD(P)H, Collagen, and Riboflavin, as well as aromatic amino acids including tyrosine, tryptophan, phenylalanine. These compounds absorb in UV to Blue range (355-488 nm), and emit in the Blue to Green range (350-550 nm). The consequence of this autofluorescence is the loss of signal resolution in these light ranges and a decrease in signal sensitivity. Autofluorescence typically increases with cell size. Larger cells have more autofluorescence than small…

Dynamic range is the total range of fluorescent values obtained from a particular flow cytometry assay. It is defined as the ratio of the largest possible fluorescent signal to the smallest possible fluorescent signal. The dynamic range can vary based on the application. For example, a cell cycle assay may have a dynamic range of only 1000 fluorescence units. Surface staining against CD3 may have a dynamic range of 10,000. There is some debate as to the largest dynamic range required for flow cytometry, with some estimates putting the largest required dynamic range at about 3.5 and others arguing for…

Sheath fluid is the solution that runs in a flow cytometer.  Once the sheath fluid is running at laminar flow, the cells are injected into the center of the stream, at a slightly higher pressure.  The principles of hydrodynamic focusing cause the cells to align, single file in the direction of flow. Depending on experimental needs, different formulations of sheath fluid can be used. Many labs purchase pre-mixed phosphate-buffered saline from Leinco Technologies. Some researchers use Hepes-buffered saline.  This is particularly useful for high-pressure cell sorting as Hepes controls pH better at high pressure than phosphate buffers do. Finally, since…

In flow cytometry, cells, in suspension are moved from the tube to the interrogation point and finally into the waste (or to be sorted, but that is a different story).  To do this, the fluidics components of the flow cytometry are required. The fluidics are comprised of a running (or Sheath) fluid, that runs through the system in laminar flow.  The movement of this sheath can be achieved by several mechanisms, the most common method using pressure provided by pumps. The second component of the fluidics is the sample injection port (SIP).  This is where the sample is pushed through…

Differential pressure based flow cytometers currently dominate the market. These systems have two pressure regulators. The first is at a constant pressure that sets how fast the fluids runs at. The second is regulated by the investigator (like as shown on this LSR-II control panel). As the sample pressure goes from low, to medium, to high, the pressure on the sample increases. This results in the volume of the sample increasing (from ~15 ml/min to ~60 ml/min). The difference between the sample pressure and the sheath pressure is the differential pressure. This controls the width of the core stream and…

A Jablonski diagram illustrates the electronic states of a molecule as well as the transitions between them. These states are arranged vertically by energy and grouped horizontally by spin multiplicity. Nonradiative transitions are indicated by straight arrows and radiative transitions by squiggly arrows. The vibrational states of each electronic state are indicated with parallel horizontal lines. For flow cytometry, its important to note that the energy of the emission is usually less than that of the absorption. As such, fluorescence normally occurs at lower energies or longer wavelengths.